Project Summary Fertility depends on successful fertilization and early development, processes that occur in the oviduct. Common therapies for human infertility, such as in vitro fertilization and intracytoplasmic sperm injection, are expensive and increase risks of a variety of problems. More knowledge of how the oviduct interacts with sperm, the cumulus-oocyte complex (COC) and the developing embryo may improve fertility and reduce the need for therapies. The oviduct serves as a reservoir for sperm, after semen deposition and before fertilization. Binding to the oviduct maintains sperm viability and suppresses motility. Sperm are released to move to the upper oviduct (ampulla) to fertilize oocytes. There are many gaps in this model of sperm-oviduct interaction. Our studies have begun to fill some of these gaps. We have used a glycomic approach to screen 377 glycans and found that all glycans with affinity for porcine sperm have either of two motifs, sulfated Lewis X trisaccharide or branched 6-sialylated complex glycans. We have identified two candidate receptors for both glycans on the sperm membrane, PKDREJ and ADAM5. Notably, mouse sperm deficient in PKDREJ and other ADAMs do not accumulate beyond the utero-tubal junction, but it is not known if this is due to a problem in binding and retention in the oviduct. Remarkably, if these glycans are immobilized on beads, they can extend sperm lifespan, much like binding to oviduct cells prolongs the lifespan of sperm. Finally, we found that cumulus-oocyte complexes (COCs) secrete molecules that signal sperm release from the lower oviduct so they can move toward the site of fertilization. The Specific Aims will provide a mechanistic understanding of how sperm bind the oviduct, how binding prolongs sperm lifespan and how sperm are released from the oviduct by the COC. Aim 1: To determine the function of PKDREJ and ADAM5 in sperm by blocking each protein and mutating each gene in swine. Sperm from pigs that are deficient in each of these proteins will be examined to determine if their ability to bind oviduct cells and their fertility is affected. Aim 2. To determine if sperm binding to immobilized oviduct glycans suppresses ROS production, conserves ATP, and stabilizes the plasma membrane, which extends sperm lifespan and fertility. Sperm bound to immobilized glycans will be examined to ascertain what changes adhesion induces. The second group of experiments will determine which changes are sufficient to prolong lifespan by adding agents that scavenge ROS and stabilize the plasma membrane. Aim 3. To determine how the cumulus-oocyte complex signals release of sperm from oviduct cells and glycans. We will examine whether progesterone, prostaglandins, or proteins that may be secreted from COCs might induce porcine sperm release from oviduct cells and immobilized glycans. The completion of these Specific Aims will elucidate how sperm bind to the oviduct, are stored in the oviduct and are released in response to the cumulus-oocyte complex. This fundamental information could be used to develop simpler, safer and less costly assisted reproductive technologies.